Aspartate Transcarbamoylase Molecules Lacking One Regulatory Subunit

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A BSTR ACTThe lack of knowledge of the threedimensional structure of the trimeric, catalytic (C) subunit of aspartate transcarbamoylase (ATCase) has impeded understanding of the allosteric regulation of this enzyme and left unresolved the mechanism by which the active, unregulated C trimers are inactivated on incorporation into the unliganded (taut or T state) holoenzyme. Surprisingly, the isolated C trimer, based on the 1.9-Å crystal structure reported here, resembles more closely the trimers in the T state enzyme than in the holoenzyme:bisubstrate-analog complex, which has been considered as the active, relaxed (R) state enzyme. Unlike the C trimer in either the T state or bisubstrate-analogbound holoenzyme, the isolated C trimer lacks 3-fold symmetry, and the active sites are partially disordered. The f lexibility of the C trimer, contrasted to the highly constrained T state ATCase, suggests that regulation of the holoenzyme involves modulating the potential for conformational changes essential for catalysis. Large differences in structure between the active C trimer and the holoenzyme:bisubstrate-analog complex call into question the view that this complex represents the activated R state of ATCase.Integration of metabolic pathways and tight control of protein functions require allosteric regulation, whose hallmark is functional cooperativity. In their pioneering paper aimed at accounting for the properties of allosteric proteins, such as hemoglobin and regulatory enzymes, Monod, Wyman, and Changeux (1) postulated that these oligomeric proteins exist in two (or more) conformational states and that ligands control the equilibrium between a low-affinity or less active, taut (T state) form and a high-affinity or more active, relaxed (R state) quaternary structure. This two-state model has been shown to account for both the homotropic cooperativity and the heterotropic effects (inhibition and activation) exhibited by Escherichia coli aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase, carbamoyl phosphate:L-aspartate carbamoyltransferase; EC 2.1.3.2), which catalyzes the first committed step in pyrimidine biosynthesis (2-4). In addition, physical chemical measurements of the change in quaternary structure and catalytic activation promoted by substoichiometric amounts of the bisubstrate analog (5), N-(phosphonacetyl)-L-aspartate (PALA), were interpreted readily in terms of the model (2).In parallel with tests of the two-state model based on enzyme kinetics and solution studies of the ligand-promoted global conformational changes in ATCase, Lipscomb and his colleagues (6-9) conducted detailed crystallographic investigations of the wild-type enzyme and various mutant forms. The extensive data include high-resolution structures of unliganded ATCase (8), PDB ID 6atl, and the PALA-liganded enzyme (9), PDB ID 8atc. Large conformational differences between the free and PALA-bound forms of the holoenzyme were proposed to define the structural differences between the T and R states.Despite the extensive kn...

Section: Resultsmentioning

confidence: 99%

Assessment of the allosteric mechanism of aspartate transcarbamoylase based on the crystalline structure of the unregulated catalytic subunit

Beernink

Endrizzi

Alber

et al. 1999

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“…Electrophoresis experiments on purified ATCase554 showed a small amount of a more rapidly migrating component that was analogous to the ATCasewtlike complex lacking one regulatory subunit (29).…”

Section: Methodsmentioning

confidence: 99%

Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation.

Schachman¹,

Pauza²,

Navre³

et al. 1984

Recent genetic studies of the pyrB locus of Escherichia coli resulted in the characterization of 29 mutant strains harboring defects in the structural gene that encodes the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). Three alleles, pyrB5S4, pyrB730, and pyrB748, have been cloned, and their nucleotide sequences have been determined along with that of the wild-type pyrBI operon in order to locate the sites of the alterations in the catalytic chains. Missense mutation pyrB554 leads to replacement of serine-52 by phenylalanine, and the inactive mutant enzyme has properties similar to those of wild-type aspartate transcarbamoylase. The amber mutation pyrB730 results in unstable truncated polypeptide chains 27 amino acids shorter than wild-type chains. Deletion mutation pyrB748 causes the removal of 181 amino acids. Combining these results with knowledge of the crystallographic structure of the wild-type enzyme provides a basis for tentative structural mechanisms for the observed complementation behavior of the mutant proteins.Despite extensive research on the complementation of inactive mutants of enzymes such as fB-galactosidase, glutamic dehydrogenase, and tryptophan synthetase (1-4), we do not yet understand how enzyme activity is restored through noncovalent interaction between defective proteins. Many precisely mapped mutants of these enzymes are available, but because x-ray evidence regarding the tertiary and quaternary structure of the proteins is lacking, it is not possible to describe in structural terms how active complexes are formed from different monomers derived from inactive mutants. Aspartate transcarbamoylase (ATCase; aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli affords an opportunity to analyze complementation in terms of protein structure because many inactive mutants of the enzyme have become available as a result of the development of a positive selection technique (5). Their in vivo complementation behavior has been determined (6), and the three-dimensional structure of the wild-type enzyme (ATCase,,) is known from crystallographic studies (7,8). The results presented here on four inactive mutants of ATCase represent the first phase of an attempt to account for the complementation of these missense, chain-termination, and deletion mutants.In a recent genetic analysis of the pyrB locus of E. coli, Jenness and Schachman (6) characterized 29 mutant strains harboring defects in the structural gene pyrB, which encodes the catalytic chains of ATCase. Interallelic complementation experiments showed that the mutants can be grouped into four complementation units that divide the fine-structure map into distinct segments (6). Those defining the a and 8 units are missense mutants (6) with amino acid substitutions located, respectively, in the polar and equatorial structural domains formed by the folded catalytic chains (7,8). The mutants comprising the 8 ...

“…Free C subunits are devoid of cooperativity and inhibition (2,6,14) as is the species CR3 (15), which is transiently observed only in the presence of a large excess of R (16). In contrast, the stable R-deficient species, C2R2 (17,18), exhibits both homotropic and heterotropic effects, although their extent is reduced compared to the native enzyme (18).…”

mentioning

confidence: 98%

Cooperative Interactions in Aspartate Transcarbamoylase. 1. Hybrids Composed of Native and Chemically Inactivated Catalytic Polypeptide Chains

Gibbons¹,

Yang²,

Schachman³

1974

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Several types of subunit interactions are implicated in stabilizing ATCase as an oligomer (6) composed of six catalytic (c) and six regulatory (r) polypeptide chains (7-9) organized as two catalytic (C) and three regulatory (R) subunits (9-12). Much evidence (13) has been accumulated in support of a model for ATCase as a complex of two C trimers bridged by three R dimers, i.e., C2R3. In this structure there are six c: c bonding domains linking the c chains in each of the two C trimers; three r:r bonding domains linking the r chains in the three R dimers, and six c: r domains linking the c and r chains (12, 13). Free C subunits are devoid of cooperativity and inhibition (2,6,14) as is the species CR3 (15), which is transiently observed only in the presence of a large excess of R (16). In contrast, the stable R-deficient species, C2R2 (17, 18), exhibits both homotropic and heterotropic effects, although their extent is reduced compared to the native enzyme (18).One systematic approach to an understanding of the structural requirements for cooperativity and feedback inhibition in ATCase involves the construction of hybrid molecules containing modified subunits (9,10). Can cooperativity and inhibition be obtained with hybrids containing one active (native) C and one inactive C and three R subunits? Would a hybrid containing active and inactive c chains in each C subunit (along with three native R subunits) exhibit allosteric behavior? As shown below, both types of ATCase-like molecules have been constructed, and they exhibit kinetic and physical properties similar to those of the native enzyme.Inactivation of the C subunits was achieved specifically by reaction of the protein with pyridoxyl 5'-phosphate followed by reduction of the Schiff base with NaBH4 (19). Since reconstituted ATCase-like molecules containing native and pyridoxylated chains did not differ sufficiently from native enzyme in electrophoretic and chromatographic behavior to permit isolation of the desired species, we introduced additional charged groups by reacting the inactive derivative, Cp, with the "reversible" anhydride, THPA (20